We propose to use a novel murine leukemia virus (MuLV)-based retroviral display system that we have recently developed (mss in prep) to improve our HIV-1 humoral vaccine candidate, which is based on the V1V2 domain of gpl20. We have recently obtained evidence indicating that the V1V2 domain of the HIV-1 envelope protein contains highly conserved epitopes that mediate potent neutralization of primary HIV-1. Our subunit vaccine candidate is a fusion protein that expresses the isolated V1V2 domain of gpl20 from a V2 consensus North American clade B isolate in the absence of other HIV sequences, using the N-terminal domain of the MuLV envelope protein to promote proper folding and secretion. Immunization of rats or macaques with this V1V2 fusion protein induces antibodies that have potent neutralizing activity for all primary virus isolates tested, including representatives of clades B, C, D and E. However, these antibodies are present in the vaccine sera at low concentration compared to antibodies against less conserved, non- neutralizing epitopes. Not only are these non-neutralizing epitopes immunodominant, but our recent evidence indicates that antibodies with enhancing activity are also induced. We will isolate variant V1V2 sequences that retain the neutralization epitopes but no longer present the non-neutralization epitopes by enrichment from random libraries using our novel MuLV retroviral particle display system. Since the desired epitopes are glycan-dependent conformational structures, standard phage display systems cannot be used for this purpose. Available antibodies against either conserved neutralization epitopes or non-neutralization epitopes will be used to separate MuLV virions bearing variants of V1V2 that express the desired epitopes from those that do not. This will involve a negative enrichment step to remove V1V2 sequence variants that express non-neutralizing epitopes followed by a positive enrichment step to require that the V1V2 variants isolated do express the potent neutralization targets in V1V2. Once sequences with improved immunoreactivity are identified, they will be expressed in our standard soluble V1V2 fusion protein system for further evaluation as immunogens. This novel methodology promises to advance our development of a V1V2 domain-targeted subunit vaccine that can induce a protective humoral response against HIV-1.